The inelastic scattering of photons is called the Raman effect, where Rayleigh scattering refers to elastic scattering. The measurement and analysis of the signals (photons) arising from the Raman effect is called Raman spectroscopy, see D. A. Long, “The Raman effect, unified treatment of the theory of Raman scattering by molecules”, John Wiley & Sons Ltd., Chichester, 2002.
A measurement device for this purpose is termed a Raman spectrometer and can be used for studying vibrational, rotational, and/or other low-frequency modes in a system, see for instance W. Demtröder, “Laser Spectroscopy”, Springer, Berlin, 2002, and “Practical Raman spectrometry” by D. J. Gardiner, Springer-Verlag, 1989.
A Raman spectrometer commonly provides a spectrum, i.e. a Raman spectrum, of intensities associated with frequency shifts with respect to incident monochromatic radiation resulting from the Raman effect. Since vibrational information is specific to the chemical bonds of molecules, the Raman spectrum provides a fingerprint by which the molecules (hereinafter referred to as analytes) can be identified. In addition, Raman spectroscopy is a potentially quantitative technique and dynamic measurements of chemical processes and interactions are possible. These features make it a useful tool in chemistry.
An example of a Raman spectrometer can be found in the international publication WO 2005/111559. A Raman spectrometer comprises a light source to illuminate a sample usually with monochromatic light, and a detector to detect Raman scattering of the illuminated sample. Optical systems are provided to direct light from the light source to the sample, to collect the scattered light, and to direct the collected scattered light to the detector.
Monochromatic light refers to electromagnetic radiation of a single wavelength. However, in practice, no real source of electromagnetic radiation is purely monochromatic. Therefore, monochromatic light also refers to sources that have a narrow range of wavelengths, such as lasers in which the narrow range of wavelengths is sometimes also referred to as spectral line width. It is noted here that the term light not necessarily refers to radiation having a wavelength in the visible range. Also wavelengths outside this range, such as infrared light, can be used. It is stated that the phenomenon of Raman scattered light is useful in spectroscopy applications for studying qualities and quantities of physical properties and processes, including identification of chemical properties, compositions, and structure in a sample.
The main problem with the acquisition of a Raman spectrum is that Raman scattering is relatively weak compared to elastic Rayleigh scattering, as a result of which the detection limit of this measurement technique is relatively poor.
The detection limit is the lowest quantity of an analyte in a sample to be measured that can be distinguished from the absence of that analyte.
An approach to improve the detection limit is filtering out the Rayleigh scattering using an appropriate filter. However, even when all Rayleigh scattering would be filtered out, the detection limit is often too poor to allow the measurement of small quantities of the analyte.
To overcome this drawback Surface Enhanced Raman Spectroscopy was developed. Surface Enhanced Raman Spectroscopy, or Surface Enhanced Raman Scattering, often abbreviated SERS, is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough surfaces. The principle of using a roughened SERS active surface (hereinafter referred to as ‘active surface’) to enhance Raman scattering is known to a person skilled in the art. SERS is a technique used to improve the detection limit for Raman measurements, commonly by deliberately targeting the molecule, i.e. the substance or analyte of interest. Targeting the molecule can be done directly by the active surface, wherein the preference of the active surface to adsorb or bond with specific substances or parts thereof is used to enrich the analyte in close proximity of the active surface. Targeting can also be done via an intermediate layer which is covering the active substrate and has a preference to adsorb or bond with specific substances or parts thereof to get the analyte in close proximity of the active surface. It is also possible that the intermediate layer does target the analyte but not for the specific purpose to get it into close proximity. In such occasions when the analyte adheres to or bonds with the intermediate layer, properties of the intermediate layer change. As the intermediate layer itself is in close proximity of the active surface, the change in properties can be measured, thereby indirectly detecting the analyte.
An advantage of targeting the molecules may be that the concentration of the substance to be measured is increased near the active surface with respect to the concentration in the analyte, and thereby also increases the enhanced Raman signal due to the substance.
By getting the substances as close to the active surface as possible, i.e. directly or indirectly, the enhancement factor of the active surface may be such that even single molecules can be detected under specific conditions. However, the technique can not be used to perform subsequent measurements of substances, as the bonding or adsorption of the substances in SERS is generally irreversible, so that a measurement will influence and/or foul a next measurement. Sensor elements in SERS are thus used for so-called one-shot measurements only. Due to this feature, a disadvantage of this technique is that it does not allow dynamic measurements, thereby limiting the possibility of calibration and quantitative measurements.
Further techniques have been developed to enhance the signal. One such technique has been to enhance the concentration of the analyte close to the surface that is being illuminated. An example therefore is to apply the so-called drop and drying technique which involves dropping or pipetting small volumes, e.g. about 1-20 μl, of an analyte solutions onto an active surface, e.g., a gold active surface, and allowing the analyte to dry. By this method remaining analyte after drying is provided as close to the active surface as possible. In addition, the gold tends to have a great affinity to the analyte, binding it firmly. Another example has been described in U.S. Pat. No. 5,721,102. This document describes a Raman spectrometer comprising a light source, an active substrate containing the analyte, and a detector. The active substrate was prepared by depositing a layer of silver with a thickness of 100 nm onto a glass strip. The silver layer was coated by a layer of e.g., silica or an organic polymer, wherein an oligonucleotide of known sequence was immobilised. The known oligonucleotide was complementary to the target oligonucleotide, which is the analyte. The analyte and the known oligonucleotide are allowed to hybridise, so that an enhanced concentration of the analyte is present at the active substrate. In US 2006/0147927 a similar technique is described; polynucleotides are attached to a surface having silver colloids and/or silver islands. The polynucleotides are complementary to a target polynucleotide sequence. The target nucleotide sequence is added to the surface and the target is allowed to hybridise.
U.S. Pat. No. 5,266,498 discloses a SERS method wherein an active surface is provided with a binding member that is bound to the active surface. The binding member has an affinity for the analyte or an indicator reagent. Upon binding of the analyte the change in the Raman signal of the binding member facilitates the detection of the analyte. In US 2005/0219509 a Raman technique is described in which the active surface and the analyte are associated with each other. Subsequently they are surrounded by an encapsulant. Such encapsulation ensures that only the Raman scattering of the analytes on the active surface will be detected.
The above-described techniques have the advantage that they concentrate the molecules to be detected in close proximity to the active surface. However, these techniques have the disadvantage that each measurement must be conducted on a separate active substrate, which adds to costs and effort. Further, these techniques render quantitative determinations very cumbersome. Moreover, these techniques cannot be used in an on-line measurement or in a dynamic determination in order to follow processes. Finally, due to their specificity they may not be suitable for the detection of unknown compounds.
Hence, whereas conventional Raman spectroscopy has the disadvantage of a weak signal, the above SERS techniques have the disadvantages that quantitative analyses and dynamic measurements are virtually impossible.